title: Mass spectrometry-based comparative sequencing to detect ganciclovir resistance in the UL97 gene of human cytomegalovirus.
abstract: BACKGROUND
  Persistent infections with herpesviruses such as human cytomegalovirus (HCMV) frequently occur after solid organ or stem cell transplantation, and are due to either failure of the host to immunologically control the virus or emerging resistance of the virus to the antiviral drug(s) used. Antiviral therapy can be guided by viral drug susceptibility testing based on screening for known resistance-inducing mutations in the viral genome. Mass spectrometry-based comparative sequence analysis (MSCSA) might be advantageous for this purpose because of its suitability for semi-automation.
  
  
  OBJECTIVE
  The applicability of MSCSA to detect sequence polymorphisms and drug resistance-inducing mutations in the HCMV genome was investigated.
  
  
  METHODS
  We analyzed the 3' part of the HCMV UL97 gene, which encodes the kinase that is activated by the commonly used anti-HCMV drug ganciclovir. Sequences obtained by MSCSA of material from HCMV-infected patients (43 samples) and the HCMV type strain were compared to conventional cycle sequencing results.
  
  
  RESULTS
  In 94.1% of all samples the results obtained by MSCSA of the UL97 gene were identical to those from conventional cycle sequencing. The threshold to detect mutant sequences in a mixture with wild-type material was 20% using either technique. Furthermore, MSCSA was successfully applied to study the development of drug resistance in a patient who developed encephalitis due to ganciclovir-resistant HCMV.
  
  
  CONCLUSIONS
  MSCSA was found to be equally accurate compared to conventional cycle sequencing in the analysis of UL97 of HCMV.
authors.affiliation: Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. c.c.posthuma@lumc.nl
authors.forename: Clara C
authors.initials: CC
authors.lastname: Posthuma
authors.forename: Martha T
authors.initials: MT
authors.lastname: van der Beek
authors.forename: Caroline S
authors.initials: CS
authors.lastname: van der Blij-de Brouwer
authors.forename: Pim L J
authors.initials: PL
authors.lastname: van der Heiden
authors.forename: Erik W A
authors.initials: EW
authors.lastname: Marijt
authors.forename: Willy J M
authors.initials: WJ
authors.lastname: Spaan
authors.forename: Eric C J
authors.initials: EC
authors.lastname: Claas
authors.forename: Christa
authors.initials: C
authors.lastname: Nederstigt
authors.forename: Ann C T M
authors.initials: AC
authors.lastname: Vossen
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Snijder
authors.forename: Aloys C M
authors.initials: AC
authors.lastname: Kroes
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001483:Base Sequence; D003587:Cytomegalovirus / Q000187:drug effects* / Q000235:genetics / Q000472:pathogenicity; D003586:Cytomegalovirus Infections / Q000188:drug therapy / Q000821:virology*; D004252:DNA Mutational Analysis / Q000379:methods; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics; D005260:Female; D015774:Ganciclovir / Q000494:pharmacology* / Q000627:therapeutic use; D005838:Genotype; D006801:Humans; D008875:Middle Aged; D008969:Molecular Sequence Data; D058889:Molecular Typing; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D000998:Antiviral Agents; D004279:DNA, Viral; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase; D015774:Ganciclovir